Osamu Shimozato

Perforin-dependent NK cell cytotoxicity is sufficient for anti-metastatic effect of IL-12

IL‐12 exerts a potent anti‐tumor effect, which is possibly mediated by multiple mechanisms including activation of NK and NKT cells, induction of cytotoxic T lymphocytes, and inhibition of angiogenesis. In the present study, we characterized the cytotoxic effector cells and mechanisms responsible for the anti‐metastatic effect of IL‐12. Administration of IL‐12 had a comparable inhibitory effect on experimental lung metastasis of B16 melanoma cells in wild‐type C57BL / 6 mice and RAG‐2 − / − mice that lack T and NKT cells, which was abolished by depletion of NK cells. Cytotoxic activity of liver and splenic mononuclear cells against B16 was induced by IL‐12 administration in RAG‐2 − / − mice at a level comparable to that in wild‐type mice, which was also abolished by depletion of NK cells. Moreover, the anti‐metastatic effect of IL‐12 was abrogated by perforin deficiency, but not by Fas ligand deficiency, in association with a lack of IL‐12‐induced cytotoxic activity of liver and splenic mononuclear cells against B16. These results suggest that perforin‐dependent cytotoxicity of IL‐12‐activated NK cells is sufficient for the anti‐metastatic effect of IL‐12.

Expression of IL-27 in murine carcinoma cells produces antitumor effects and induces protective immunity in inoculated host animals.

A novel cytokine interleukin‐27 (IL‐27), composed of p28 and Epstein‐Barr virus‐induced gene 3 (EBI3), is produced from activated dendritic cells and is involved in an early phase of T‐helper type I differentiation. We examined whether Colon 26 murine colon carcinoma cells that were retrovirally transduced with the p28‐linked EBI3 gene (Colon 26/IL‐27) could produce antitumor effects in inoculated mice. Although proliferation in vitro of Colon 26/IL‐27 cells was not different from that of parent cells, syngeneic BALB/c mice rejected Colon 26/IL‐27 tumors inoculated and subsequently acquired tumor‐specific protective immunity. In contrast, mice inoculated with Colon 26 cells transduced with either the p28 or EBI3 gene developed tumors and survival of the mice remained the same as that of the mice inoculated with parent cells. Syngeneic nude mice developed Colon 26/IL‐27 tumors, but the growth was retarded compared to that of parent tumors. Depletion of natural killer cells from nude mice with anti‐asialo GM1 antibody diminished the growth retardation of Colon 26/IL‐27 tumors. Survival of severe combined immunodeficient mice that received subcutaneous inoculation of Colon 26/IL‐27 cells was not different from that of the immunodeficient mice inoculated with parent cells. Interferon‐γ was produced from CD4+ and CD8+ T, and natural killer cells of the mice that rejected Colon 26/IL‐27 tumors and cytotoxic activity against Colon 26 cells were also detected from the mice. These data collectively suggest that expressed IL‐27 in tumors produces T cell‐dependent and‐independent antitumor effects and is a possible therapeutic strategy for cancer. ©2005 Wiley‐Liss, Inc.

CD133 suppresses neuroblastoma cell differentiation via signal pathway modification

CD133 (prominin-1) is a transmembrane glycoprotein expressed on the surface of normal and cancer stem cells (tumor-initiating cells), progenitor cells, rod photoreceptor cells and a variety of epithelial cells. Although CD133 is widely used as a marker of various somatic and putative cancer stem cells, its contribution to the fundamental properties of cancer cells, such as tumorigenesis and differentiation, remains to be elucidated. In the present report, we found that CD133 was expressed in several neuroblastoma (NB) cell lines/tumor samples. Intriguingly, CD133 repressed NB cell differentiation, for example neurite extension and the expression of differentiation marker proteins, and was decreased by several differentiation stimuli, but accelerated cell proliferation, anchorage-independent colony formation and in vivo tumor formation of NB cells. NB cell line and primary tumor-sphere experiments indicated that the molecular mechanism of CD133-related differentiation suppression in NB was in part dependent on neurotrophic receptor RET tyrosine kinase regulation. RET transcription was suppressed by CD133 in NB cells and glial cell line-derived neurotrophic factor treatment failed to induce RET in CD133-expressing cells; RET overexpression rescued CD133-related inhibition of neurite elongation. Of note, CD133-related NB cell differentiation and RET repression were mainly dependent on p38MAPK and PI3K/Akt pathways. Furthermore, CD133 has a function in growth and RET expression in NB cell line- and primary tumor cell-derived tumor spheres. To the best of our knowledge, this is the first report of the function of CD133 in cancer cells and our findings may be applied to improve differentiation induction therapy for NB patients.

Expression of the interleukin-21 gene in murine colon carcinoma cells generates systemic immunity in the inoculated hosts

Interleukin-21 (IL-21) is a novel cytokine that can induce proliferation of activated T cells and maturation of natural killer (NK) cells. We therefore examined whether expression of the IL-21 gene in tumor cells could generate antitumor responses. Murine colon carcinoma Colon 26 cells that were transduced with the mouse IL-21 gene (Colon 26/IL-21) were rejected in syngeneic mice and the mice subsequently acquired protective immunity. The growth of Colon 26/IL-21 tumors developed in nude mice was retarded compared with that of parent tumors, and this growth suppression was not observed in nude mice that were treated with anti-asialo GM1 antibody. Spleen cells from the mice that had rejected Colon 26/IL-21 cells showed cytotoxic activity to Colon 26 but not to irrelevant tumor cells, and produced larger amounts of interferon-γ upon stimulation with irradiated Colon 26 cells. Spleen cells from Colon 26/IL-21-tumor- but not parent-tumor-bearing mice had lytic activity to YAC-1 cells. These data suggest that expression of IL-21 in tumors induces T- and NK-cell-dependent antitumor effects.

Transduction of the IL-21 and IL-23 genes in human pancreatic carcinoma cells produces natural killer cell-dependent and -independent antitumor effects

We examined whether novel cytokines, interleukin (IL)-21 and IL-23, that were expressed in tumors could produce antitumor effects in the inoculated mice. Human pancreatic cancer AsPC-1 cells were retrovirally transduced with murine IL-21 or IL-23 (p19-linked p40) gene (AsPC-1/IL-21, AsPC-1/IL-23) and were injected into nude or severe combined immunodeficiency (SCID) mice. Although the proliferation in vitro of the transduced cells remained the same as that of parent cells, growth of AsPC-1/IL-21 and AsPC-1/IL-23 tumors developed in nude mice was retarded compared with that of parent tumors. Treatment of nude mice with anti-asialo GM1 antibody temporally abrogated the growth retardation of AsPC-1/IL-21, but not AsPC-1/IL-23 tumors; however, the growth of AsPC-1/IL-21 tumors came to be retarded thereafter with the regeneration of natural killer (NK) cells. The growth of AsPC-1/IL-21 tumors developed in SCID mice was also retarded compared with parent tumors and the growth retardation was abrogated by treatment with anti-asialo GM1 antibody. The growth of AsPC-1/IL-23 tumors in SCID mice was not different from that of parent tumors. Cytotoxic activity and secretion of interferon-γ in response to AsPC-1 cells were induced in spleen cells of the mice bearing AsPC-1/IL-21 or AsPC-1/IL-23 tumors. When nude mice were injected with a mixed population of AsPC-1/IL-21 and AsPC-1/IL-23 cells, no synergistic effects were observed. These data collectively suggest that expression of IL-21 and IL-23 in tumors can produce NK cell-dependent and -independent antitumor effects in an αβ T cell-defective condition, respectively.

Induction of systemic immunity by expression of interleukin-23 in murine colon carcinoma cells

Interleukin‐23 (IL‐23), a novel cytokine composed of a newly identified p19 molecule and the p40 subunit of IL‐12, can stimulate the proliferation in vitro of memory T cells. We examined whether Colon 26 murine colon carcinoma cells that were retrovirally transduced with the p19‐linked p40 gene (Colon 26/IL‐23) could produce antitumor effects in inoculated mice. The growth of Colon 26/IL‐23 tumors developed in immunocompetent mice was significantly retarded and the tumors disappeared thereafter. Spleen cells from the mice that received Colon 26/IL‐23 cells produced significant amounts of interferon‐γ, when they were cultured with irradiated Colon 26 but not irrelevant cells. Depletion of CD8+ T cells suppressed the production of interferon‐γ. The mice that had rejected Colon 26/IL‐23 tumors were resistant to subsequent challenge of parent but not irrelevant tumor cells. Colon 26/IL‐23 tumors were not rejected in nude mice but the growth was retarded compared to parent tumors. Treatment of nude mice with anti‐asialo GM1 antibody did not influence the growth of Colon 26/IL‐23 tumors. These data suggest that expression of IL‐23 in tumors produces T cell‐dependent antitumor effects and induces systemic immunity.

The secreted form of the p40 subunit of interleukin (IL)‐12 inhibits IL‐23 functions and abrogates IL‐23‐mediated antitumour effects

Interleukin (IL)‐23 is a heterodimeric cytokine consisting of a novel p19 molecule and the p40 subunit of IL‐12. Since secreted p40 can act as an antagonist for IL‐12, we investigated whether p40 also inhibited IL‐23‐mediated immunological functions. p40 did not induce interferon (IFN)‐γ or IL‐17 production from splenocytes but impaired IL‐23‐induced cytokine production by competitive binding to the IL‐23 receptors. Furthermore, a mixed population of murine colon carcinoma Colon 26 cells transduced with the p40 gene and those transduced with the IL‐23 gene developed tumours in syngenic mice, whereas the IL‐23‐expressing Colon 26 cells were completely rejected. p40 also suppressed IFN‐γ production of antigen‐stimulated splenocytes and IL‐23‐mediated cytotoxic T‐lymphocyte activities in the mice that rejected Colon 26 cells expressing IL‐23. p40 can thereby antagonize IL‐23 and is a possible therapeutic agent for suppression of IL‐23 functions.

Expression of tumour necrosis factor (TNF) ligand superfamily co-stimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis caused by Coxsackievirus B3.

Antigen‐specific T‐cells infiltrate the heart and play an important role in the myocardial damage involved in acute myocarditis and dilated cardiomyopathy. To investigate the roles of the co‐stimulatory molecules CD30/CD30L, CD27/CD27L, OX40/OX40L, and 4‐1BB/4‐1BBL, which belong to the tumor necrosis factor (TNF) receptor/ligand superfamily, in the development of acute viral myocarditis, the expression of these molecules was first analysed in the hearts of mice with acute myocarditis induced by Coxsackievirus B3 (CVB3) in vivo. Secondly, the induction of these molecules was evaluated on cultured cardiac myocytes treated with interferon (IFN)‐γ and the interleukin (IL)‐6 production by cultured cardiac myocytes was analysed by stimulation with monoclonal antibodies (MAbs) against these molecules in vitro. Thirdly, the effects of in vivo administration of anti‐CD30L, anti‐CD27L, anti‐OX40L, or anti‐4‐1BBL MAb on the development of acute viral myocarditis were examined. CVB3‐induced myocarditis resulted in the induction of CD30L and 4‐1BBL on the surface of cardiac myocytes, confirmed by treatment with IFN‐γ in vitro. CD27L and OX40L were constitutively expressed on cardiac myocytes in vivo and in vitro. Anti‐CD30L and anti‐4‐1BBL MAbs stimulated IL‐6 production by cardiac myocytes in vitro. Furthermore, in vivo anti‐4‐1BBL MAb treatment significantly decreased the myocardial inflammation, whereas the other MAbs did not. These findings suggest that TNF ligand superfamily co‐stimulatory molecules, especially 4‐1BBL, play an important role in the development of acute viral myocarditis and raise the possibility that immunotherapy with anti‐4‐1BBL MAb may be of benefit in acute viral myocarditis. Copyright

Impairment of Bleomycin-Induced Lung Fibrosis in CD28-Deficient Mice

Lung fibrosis is an important pulmonary disease with a high mortality rate, but its pathophysiological mechanism has not been fully clarified. Various types of cells have been implicated in the development of lung fibrosis, including T cells. However, the contribution of functional molecules expressed on T cells to the development of lung fibrosis remains largely unknown. In this study, we determined whether costimulation via CD28 on T cells was crucial for the development of lung fibrosis by intratracheally administering bleomycin into CD28-deficient mice. Compared with wild-type mice, the CD28-deficient mice showed markedly impaired lung fibrosis after injection with low doses of bleomycin, as judged by histological changes and hydroxyproline content in the lungs. In addition, bleomycin-induced T cell infiltration into the airways and production of several cytokines and chemokines including IL-5 were also impaired in the CD28-deficient mice. Furthermore, adoptive transfer of CD28-positive T cells from wild-type mice recovered the impaired bleomycin-induced lung fibrosis in CD28-deficient mice. These findings suggest that the CD28-mediated T cell costimulation plays a critical role in the development of lung fibrosis, possibly by regulating the production of cytokines and chemokines in the lung. Thus, manipulation of the CD28-mediated costimulation could be a potential therapeutic strategy for the prevention of lung fibrosis.

The secreted form of p28 subunit of interleukin (IL)-27 inhibits biological functions of IL-27 and suppresses anti-allogeneic immune responses

Interleukin‐27 (IL‐27) is a new IL‐12‐related heterodimeric cytokine comprising a novel p28 molecule and the Epstein–Barr‐virus‐induced gene 3 (EBI3) molecules. It augments initiation of T helper type 1‐mediated immunity by enhancing the proliferation and cytokine production of T cells. In this study, we examined whether a secreted form of IL‐27 subunits would inhibit IL‐27‐mediated immunological responses. COS‐7 cells transduced with the mouse (m) p28 gene secreted a monomeric mp28 protein; however, those transduced with the mEBI3 gene did not detect a mEBI3 protein in the culture supernatants. The secreted mp28 prevented the IL‐27‐mediated signal transduction and activator of transcription 1 phosphorylation and subsequently inhibited the IL‐27‐mediated intercellular adhesion molecule‐1 induction and interferon‐γ production in CD4+ T cells. We generated mp28‐expressing murine carcinoma Colon 26 cells and inoculated a mixture of the mp28‐ and mIL‐27‐expressing Colon 26 cells into syngeneic BALB/c mice. Simultaneous production of mp28 and mIL‐27 from Colon 26 cells suppressed IL‐27‐mediated anti‐tumour effects in the mice. We examined the p28‐mediated immune suppression by inoculating mp28‐expressing myoblasts into allogeneic mice. Forced production of mp28 suppressed the allogeneic cytotoxic T‐lymphocyte induction and subsequently retarded the graft rejection. Furthermore, production of both mp28 and mp40, which inhibits the functions of IL‐12 and IL‐23, prolonged the graft survival longer than the grafts expressing either mp28 or mp40. We propose that p28 can be a regulatory subunit for IL‐27‐mediated cellular immune responses and a possible therapeutic agent to suppress unfavourable immune responses.